CBL Deletion Mutant Found in AML Patients Cause Transformation of Receptor Tyrosine Kinase Class III Expressing Cells by Activation of the AKT Pathway

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2447-2447
Author(s):  
Harald Polzer ◽  
Hanna Janke ◽  
Wolfgang Hiddemann ◽  
Dirk Eick ◽  
Karsten Spiekermann
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Deletion Mutant ◽  
Negative Regulator ◽  
Akt Pathway ◽  
Deletion Mutants ◽  
Class Iii ◽  
Akt Phosphorylation ◽  
Interaction Sites ◽  
Ligand Stimulation

Abstract Abstract 2447 We examined the oncogenic potential of CBL deletion mutant found in AML patients in cytokine receptor and receptor tyrosine kinase (RTKs) expressing cells. In addition, we analyzed the interaction sites of FLT3/CBL and the critical pathways activated by CBL deletion mutants. RTK, CBL and AKT constructs were expressed in Ba/F3 cells via a retroviral expression vector. Stable protein expression after transduction and fluorescence-activated cell sorting (FACS) was confirmed by western blotting and cellsurface-marker expression of receptors by flow cytometry. Cell Proliferation and apoptosis assays were done in presence and absence of IL-3 or receptor-ligands. Coexpression of RTK III-WT (PDGFRA, PDGFRB, FLT3, KIT) and CBL deletion mutants cause IL-3 independent and ligand dependent growth of Ba/F3 cells. RTK III-WT/CBLΔexon8 cells show a more than 10 fold hyperproliferation in response to ligand stimulation. In contrast Non-class III receptor tyrosine kinases (EGFR, EPOR, MPL, IGF1R) and CSF1R show just a very weak hyperproliferation if coexpressed with the CBL deletion mutant. Selective protein tyrosine kinase inhibitors abrogate this proliferation. In cells coexpressing RTK-III receptor and CBLΔexon8 the receptor internalization is delayed and cells were protected from apoptosis after cytokine withdrawal. Ba/F3 cells after ligand stimulation and AML cell lines coexpressing CBL deletion mutants and FLT3 show an enhanced AKT phosphorylation. The PI3K inhibitor LY294002 and the AKT inhibitor MK2206 abolish the CBL mutant mediated hyperproliferation. Furthermore, a combined pharmacological inhibition of PI3K/AKT pathway and RTK shows an additive effect. The transforming potential of the CBL mutant is completely abolished by a mutated PTB domain of CBL (G306E) and decreased by mutation of tyrosines 589 and 591 in the juxtamembrane domain of FLT3. A constitutive active AKT mutant (E17K) recapitulates the CBL deletion mutant induced phenotype in Ba/F3 cells. CBL is a selective negative regulator of class III RTK receptors and the PI3K/AKT pathway is critical for the transforming potential of the CBL oncogene. An alternative mechanism for the constitutive activation of RTKs in tumors occurs through inactivation of a negative regulator. CBL mutants mirror the phenotype of oncogenic RTK and cause an enhanced AKT phosphorylation. Targeted inhibition of FLT3 and AKT might be of therapeutic value in AML patients carrying CBL deletion mutants.Figure:Hyperproliferation of Ba/F3 cells coexpressing indicated receptors and CBL deletion mutant is quoted as X-fold of CBL wildtype coexpressing cells.Figure:. Hyperproliferation of Ba/F3 cells coexpressing indicated receptors and CBL deletion mutant is quoted as X-fold of CBL wildtype coexpressing cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Amalgam regulates the receptor tyrosine kinase pathway through Sprouty in glial cell development in the Drosophila larval brain

2020 ◽  
Vol 133 (19) ◽  
pp. jcs250837
Author(s):  
Majd M. Ariss ◽  
Alexander R. Terry ◽  
Abul B. M. M. K. Islam ◽  
Nissim Hay ◽  
Maxim V. Frolov
Keyword(s):  
Cell Proliferation ◽  
Tyrosine Kinase ◽  
Glial Cell ◽  
Receptor Tyrosine Kinase ◽  
Cell Development ◽  
Negative Regulator ◽  
Larval Brain ◽  
Adhesion Protein ◽  
Type Composition ◽  
Signaling Activation

ABSTRACTThe receptor tyrosine kinase (RTK) pathway plays an essential role in development and disease by controlling cell proliferation and differentiation. Here, we profile the Drosophila larval brain by single-cell RNA-sequencing and identify Amalgam (Ama), which encodes a cell adhesion protein of the immunoglobulin IgLON family, as regulating the RTK pathway activity during glial cell development. Depletion of Ama reduces cell proliferation, affects glial cell type composition and disrupts the blood–brain barrier (BBB), which leads to hemocyte infiltration and neuronal death. We show that Ama depletion lowers RTK activity by upregulating Sprouty (Sty), a negative regulator of the RTK pathway. Knockdown of Ama blocks oncogenic RTK signaling activation in the Drosophila glioma model and halts malignant transformation. Finally, knockdown of a human ortholog of Ama, LSAMP, results in upregulation of SPROUTY2 in glioblastoma cell lines, suggesting that the relationship between Ama and Sty is conserved.

scholarly journals Receptor tyrosine kinase activation of RhoA is mediated by AKT phosphorylation of DLC1

2017 ◽  
Vol 216 (12) ◽  
pp. 4255-4270 ◽  
Author(s):  
Brajendra K. Tripathi ◽  
Tiera Grant ◽  
Xiaolan Qian ◽  
Ming Zhou ◽  
Philipp Mertins ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Receptor Tyrosine Kinase ◽  
Rho Gtpase ◽  
Focal Adhesions ◽  
Cancer Cell Line ◽  
Suppressor Activity ◽  
Akt Phosphorylation ◽  
Transformed Cell Lines

We report several receptor tyrosine kinase (RTK) ligands increase RhoA–guanosine triphosphate (GTP) in untransformed and transformed cell lines and determine this phenomenon depends on the RTKs activating the AKT serine/threonine kinase. The increased RhoA-GTP results from AKT phosphorylating three serines (S298, S329, and S567) in the DLC1 tumor suppressor, a Rho GTPase-activating protein (RhoGAP) associated with focal adhesions. Phosphorylation of the serines, located N-terminal to the DLC1 RhoGAP domain, induces strong binding of that N-terminal region to the RhoGAP domain, converting DLC1 from an open, active dimer to a closed, inactive monomer. That binding, which interferes with the interaction of RhoA-GTP with the RhoGAP domain, reduces the hydrolysis of RhoA-GTP, the binding of other DLC1 ligands, and the colocalization of DLC1 with focal adhesions and attenuates tumor suppressor activity. DLC1 is a critical AKT target in DLC1-positive cancer because AKT inhibition has potent antitumor activity in the DLC1-positive transgenic cancer model and in a DLC1-positive cancer cell line but not in an isogenic DLC1-negative cell line.

Role and Regulation of CSF-1-Induced CSF-1 Receptor Interchain Disulfide Bonding in Receptor Activation in Macrophages

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3869-3869
Author(s):  
E. Richard Stanley ◽  
Ying Xiong ◽  
Da Song ◽  
Wenfeng Yu ◽  
Yee-Guide Yeung
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Receptor Tyrosine Kinase ◽  
Morphological Characteristics ◽  
Receptor Activation ◽  
Third Party ◽  
Mouse Bone Marrow ◽  
Pdgf Receptor ◽  
Class Iii ◽  
Disulfide Bonding

Abstract Colony stimulating factor-1 (CSF-1) is the major regulator of tissue macrophage development and function. The effects of CSF-1 are mediated by the CSF-1 receptor (CSF-1R), a class III receptor tyrosine kinase belonging to the PDGF receptor family. To study CSF-1R structure/function in macrophages using both genetic and proteomic approaches, we developed a novel, CSF-1R-deficient, mouse bone marrow macrophage (BMM) line (MacCsf1r−/−) (Yu et al., J. Leuk. Biol.84: in press, 2008). MacCsf1r−/− macrophages are maintained in GM-CSF. Retroviral expression of the wild type CSF-1R fully rescued the CSF-1-induced survival, proliferation, differentiation and morphological characteristics, which resemble those of primary BMM. We have studied CSF-1-induced covalent modifications of the receptor in MacCsf1r−/− cells. Activation of the CSF-1R involves ligand-induced receptor dimerization and trans-phosphorylation of the cytoplasmic domains. Besides tyrosine phosphorylation, CSF-1 stimulation results in disulfide bonding of the CSF-1R dimers and cysteine mutagenesis experiments revealed that specific residues in the kinase domain are involved. Inhibition of the disulfide bond formation significantly compromised CSF-1-induced CSF-1R tyrosine phosphorylation, indicating its requirement for full receptor activation. Analysis of the regulation of CSF-1-induced CSF-1R interchain disulfide bonding revealed the participation of third party molecules with additional CSF-1R post-translational modifications. The importance of ligand-induced disulfide bonding for class III receptor tyrosine kinase activation may not be restricted to the CSF-1R as interchain disulfide bonding of PDGF receptor dimers is also ligand-induced.

Phase 1 AML Study of AC220, a Potent and Selective Second Generation FLT3 Receptor Tyrosine Kinase Inhibitor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 767-767 ◽  
Author(s):  
Jorge E. Cortes ◽  
Darejan Ghirdaladze ◽  
James M. Foran ◽  
Marcel P. DeVetten ◽  
Mamia Zodelava ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Phase 1 ◽  
Cell Transplant ◽  
Class Iii ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Flt3 Mutations

Abstract Activating mutations in the receptor tyrosine kinase FLT3 are present in approximately 30% of patients (pts) with acute myeloid leukemia (AML), and they have a significantly worse prognosis than pts with wild type (WT) FLT3, suggesting that the activated kinase is a driver of the disease and a potential target for kinase inhibitor therapy. AC220 is a novel 2nd generation class III receptor tyrosine kinase (RTK) inhibitor with potent in vitro and in vivo activity in FLT3-dependent tumors. It is highly selective for WT and mutant FLT3 and several other class III RTKs, including KIT, CSF1R, RET and PDGFR. AC220 is in a first-in-human phase 1 study for relapsed or refractory AML pts, unselected for FLT3 mutations. The study has a standard 3+3 dose escalation design with 50% dose increments. AC220 is administered once daily as an oral solution for 14 days followed by a 14 day rest period (1 cycle) with a starting dose of 12 mg. Concurrently, pts are being dosed on a continuous dosing regimen starting at 200 mg/day for 28 days (1 cycle). Pts with clinical benefit may continue to receive further cycles. Currently, 52 pts have been dosed with AC220 up to 450 mg/day (10 dose cohorts). Median age was 60 yrs (range, 23 to 86 yrs), median number of prior therapies was 3 (range, 0 to 8) and 2 pts had prior allogeneic hematopoietic stem cell transplant (HSCT). Two elderly patients (age ≥ 78 yrs) unfit for induction chemotherapy were previously untreated. Fifteen patients have FLT3 mutations (12 ITD and 3 TKD), 25 are WT, and 12 are undetermined. Pts are also evaluated for PK, pFLT3, pSTAT5, FLT3 genotyping and ex vivo plasma inhibitory activity. AC220 is well tolerated and MTD has not yet been observed with either schedule. One pt had a possibly drug-related DLT in the 18 mg cohort (grade 3 CHF, although pt had a pre-existing heart condition) leading to cohort expansion, but no other cases of drug-related CHF or other DLT have been seen. Other possibly drug-related AEs (most frequently gastrointestinal events) were mild (grade ≤ 2). Response data based on investigator’s assessment are available on the first 45 pts. Responses were observed in 11 (24%) pts. Four pts achieved a complete response (CR) – 2 with incomplete platelet recovery (CRp) and 2 with incomplete platelet and neutrophil recovery (CRi), one of these pts also had complete resolution of leukemia cutis. In addition, 7 pts had partial responses (PR, defined as a decrease of ≥ 50% blasts to levels of 5%–25% in the bone marrow). Most responses (8/11, 73%) occurred after cycle 1 and one was observed after cycle 3. Median duration of response is 18 weeks (range, 4 to 26+ weeks). Three responders are FLT3 mutants (2 ITD and 1TKD), 5 are WT, 3 are undetermined. Six of the 9 non-responding pts with ITD mutations had initial rapid clearing of peripheral blasts with intermittent AC220 dosing, but subsequently progressed or had disease-related mortality. All these pts had aggressive disease and received a median of 6 prior treatment regimens (range, 3 to 8). AC220 plasma exposure is sustained between dose intervals and continues to increase in a dose-proportional manner from 12 mg to 300 mg. FLT3 phosphorylation is strongly suppressed when plasma obtained from study pts is tested ex vivo in FLT3-ITD and WT cell lines at 12 mg and 60 mg doses, respectively. Assessments of pFLT3 and pSTAT5 from treated pts’ peripheral blood are ongoing and will be presented. Encouraging preliminary efficacy results and an acceptable safety profile warrants continued evaluation of AC220 as a single agent and in combination with other therapeutics for the treatment of AML.

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